Capture assay utilising a particulate analyte

ABSTRACT

Device ( 1 ) for assaying a particulate analyte, comprising (a) a receptacle ( 2 ) for receiving a suspension ( 5 ) of particulate analyte, and containing label ( 3 ) for the particulate analyte, and (b) a porous detection material ( 4 ) that is permeable to label ( 3 ) but is impermeable to particulate analyte, the detection material ( 4 ) being arranged within receptacle ( 2 ) such that introduction of suspension ( 5 ) of particulate analyte into receptacle ( 2 ) forms liquid communication between label ( 3 ) and detection material ( 4 ). After liquid sample ( 5 ) is introduced into the receptacle ( 2 ), capillary flow through the porous detection material ( 4 ) begins. Particulate analyte cannot flow through the detection material ( 4 ) and is captured at its surface. Addition of liquid sample ( 5 ) also releases the label ( 3 ) within the receptacle ( 2 ), which is then free to flow through the detection material ( 4 ). Label ( 3 ) encounters the captured analyte and gives a signal ( 7 ). Label is prevented from running ahead of the analyte, resulting in high sensitivity.

All documents cited herein are incorporated by reference in theirentirety.

TECHNICAL FIELD

This invention relates to assay devices for measuring analytes. Inparticular, it relates to devices which capture analytes mechanicallywithin a porous material, rather than using conventional immuno-capturetechniques.

BACKGROUND ART

The format of the standard rapid test lateral flow device has remainedunchanged for around ten years. Typically, the device will comprise anitrocellulose strip. A sample is applied to an application zone, fromwhich it flows by capillary action through a zone containing avisibly-labelled antibody specific for the analyte in question. Free andbound label continue to migrate to a capture zone, where an immobilizedantibody specific for the analyte binds the analyte-label complex. Freelabel (unbound antibody) continues to migrate, leaving ananalyte-specific signal in the capture zone. These types of lateral flowdevices are disclosed in, for example, EP-A-0284232. Numerous variationsof the basic assay have been described, including those in WO92/12428,EP-A-0613005, WO97/06439, and U.S. Pat. No. 5,741,662.

In all cases, however, capture of the analyte-label complex is mediatedby an immobilized reagent, which is typically an antibody that isspecific for the analyte. This is unsatisfactory in many respects.

Firstly, manufacturing quality control is difficult. The solid phasecapture membrane is typically made from nitrocellulose, and antibodiesare applied to the membrane directly. Nitrocellulose manufacture is not,however, homogeneous. Quality control of the solid phase antibody istherefore limited to testing a statistical sample of devices from thesame, but heterogeneous, batch, and assuming that the whole batch willperform within specific tolerances. It is well known, however, thatmembranes vary considerably, even within a single batch or lot number.

Secondly, they are relatively cumbersome to manufacture. The applicationof an immobilized antibody to the strip requires a separate step fromthe application of the mobile labelled antibody. The capture antibodycan be sprayed directly onto the nitrocellulose strip, but the labelantibody has to be separately applied to fibrous material which issubsequently attached to the nitrocellulose strip, with an overlap toensure capillary flow.

Thirdly, the antibody is immobilized by spraying a solution onto amembrane. Some of the antibody does not bind to the membrane strongly,however, and some remains loosely associated with the immobilizedantibody. This semi-bound or unbound antibody can become mobile when thesolvent front passes over it, resulting in lower binding of label at thedetection zone. If the device includes a control line, this will capturethe additional label which should have been captured at the detectionzone. Tests that rely on a comparison of color intensity between controland detection lines, such as ovulation prediction kits, may thereforegive false results. Furthermore, application by spraying inevitablyleads to diffusion into the membrane, leading to a more diffuse and lessfocused detection signal.

Fourthly, the sensitivity of the devices is limited by their format.Analyte and labelled-antibody react as they migrate through themembrane, and flow rates are therefore adjusted to enable thelabelled-antibody to flow at the solvent front in order to maximize theamount of time in which the analyte-label complex can form. The complexpasses over the capture antibody for a short time, however, thusimposing constraints on the design of the test and its performancecharacteristics. The short reaction time decreases sensitivity, and alsomeans that high affinity capture antibodies are required.

Finally, the shelf-life of these test devices is often limited by thecollapse of the immobilized capture antibody onto the membrane overtime.

These shortcomings in the prior art devices are addressed byinternational patent application WO00/20866, which discloses a devicefor assaying an analyte, comprising a labelling zone, where a label canbind to the analyte, in communication with a capture zone, wherein thepore size of the capture zone is such that label which is not bound tothe analyte can migrate therethrough, whereas label which is bound tothe analyte cannot. During migration from the labelling zone to thecapture zone, therefore, unbound label can pass into and through thecapture zone, whereas bound label will be captured at the junction ofthe labelling zone and the capture zone.

A similar concept is disclosed in international patent applicationPCT/GB00/04140, which discloses a lateral flow device for assaying ananalyte, having a porous reaction zone in communication with a porousfilter zone, wherein the reaction zone contains (i) an analyte-specificlabel and (ii) a particulate carrier having an analyte-specific capturereagent immobilized thereon. The filter zone has a smaller pore sizethan that of the reaction zone, such that label that is not bound to theparticulate carrier can migrate into the filter zone, whereas label thatis bound to the particulate carrier cannot.

The main difference between these two applications is that inWO00/20866, the flow of the analyte is retarded, whereas inPCT/GB00/04140, the flow of the particulate carrier is retarded. In bothcases, however, reduced pore size is used for immobilization on thestrip, rather than using conventional immuno-capture techniques.

It has now been found that sensitivity of devices that use small poresto capture reagents can be increased by allowing direct contact betweenthe porous capture material and the analyte.

SUMMARY OF THE INVENTION

The invention provides a device for assaying a particulate analyte,comprising (a) a receptacle for receiving a suspension of theparticulate analyte, and containing a label for the particulate analyte,and (b) a porous detection material that is permeable to the label butis impermeable to the particulate analyte. The detection material isarranged within the receptacle such that introduction of the suspensionof the particulate analyte into the receptacle forms a liquidcommunication between the label and the detection material.

Compared with the devices of WO00/20866 and PCT/GB00/04140, in whichanalyte and label interact before meeting the zone with reduced poresize, the device of the present invention can capture the particulateanalyte and then allow label to bind to it. This prevents label fromrunning ahead of the analyte, resulting in much better assaysensitivity.

Typically, after a liquid sample is introduced into the receptacle,capillary flow through the porous detection material begins. Particulateanalyte cannot enter and flow through the detection material, however,so it is captured at or near its surface. The addition of the liquidsample also releases or activates the label within the receptacle, whichis then free to flow into and through the detection material. Labelencounters the captured analyte and gives a signal. Surprisingly, thesignal is discrete and sharp.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing illustrating steps of assaying using thepresent invention;

FIG. 2 is a schematic drawing illustrating alternative steps of assayingusing the present invention;

FIG. 3 is an exploded perspective view of a device for performingassaying according to the present invention; and

FIG. 4 is a perspective view of the device of FIG. 3.

DETAILED DESCRIPTION OF THE INVENTION

The Porous Detection Material

The porous detection material is permeable to label but is impermeableto the particulate analyte. This means that particulate analyte cannotenter and flow through it, but is instead captured at or near itssurface. Label can flow into and through the detection material, bindingto any captured analyte. As the concentration of analyte in a sampleincreases, the amount of label retained by the detection material alsoincreases, thus allowing semi quantitative measurement of analyte.Significantly, the analyte is not within a fibrous matrix at the pointof contact with the porous detection material (i.e., the porousdetection material has a pore size that substantially prevents theparticulate analyte from entering the detection material at the pointwhere the particulate analyte contacts the detection material).

The detection material can be made from any suitable porous materialthrough which unbound label can migrate whilst particulate analytecannot. This requirement will be reflected in the pore size of thedetection material. In one embodiment, the detection material is madefrom HDPR with a nominal pore size of between 1–100 μm, preferablybetween 15–75 μm, and more preferably between 25–50 μm. In anotherembodiment, the detection material is made from nitrocellulose, with anominal pore size of between 1–15 μm, preferably between 3–10 μm, andmore preferably with a nominal pore size between 5–8 μm (e.g. 6 μm).

As is well known to those in the art, the nominal pore size of a porousmaterial can be determined by hard particle challenge testing (i.e., bydetermining the maximum diameter of spherical particles which can passthrough the material). Alternatively, the pore size of a material may bedetermined by measuring its ‘bubble point’. The bubble point is thepressure required to force air through a (water) wet membrane, andcorrelates with the pore size as measured by particle retention(although at extremes of pressure and pore size, the correlation may beweaker). The bubble point is generally easier to measure than particleretention and is thus the preferred test when assessing pore size.

When the device of the present invention is to be used for assaying amotile analyte in particular (such as motile spermatozoa or motilebacteria), the appropriate pore size may be determined empirically byroutine testing.

The porous detection material is arranged within the receptacle suchthat introduction of a liquid suspension of analyte into the receptacleforms a communication between the label and the detection material.Before addition of the suspension, the label and the detection materialare not in liquid communication, such that label cannot enter thedetection material by capillary action. After addition of thesuspension, however, the label is activated and can migrate into andthrough the porous detection material, where it can bind to any capturedanalyte.

The porous detection material is preferably in the form of a strip.

The Particulate Analyte

The device is particularly suitable for assaying analytes such asbiological cells, which are naturally particulate. Preferred cells forassay are mammalian cells and micro-organisms.

The analyte is preferably spermatozoa. The label preferably recognizes asurface antigen which is present on the majority of a population ofspermatozoa, rather than a subset. Whilst sperm-specific antigens may beused (e.g. P34H (WO97/40836), SP-10 (WO95/29188), see alsoEP-A-0387873), ‘universal’ antigens such as CD59 may be used. It will beappreciated that, where the antigen is not sperm-specific (i.e. it isalso present on other cell types, such as CD59), the sample beinganalyzed may require treatment to remove non-spermatozoa cells.Typically, the sperm-containing sample to be analyzed will not be ‘neat’semen, but will be diluted, and possibly treated to removenon-spermatozoa cells. If ‘neat’ semen is analyzed, it will generally benecessary to use a sperm-specific label, so that non-spermatozoa cellsare not labelled. The detection material for retarding the passage ofspermatozoa is preferably a nitrocellulose membrane with a nominal poresize of between 5 μm and 8 μm. A sperm sample may be treated to separatemotile and non-motile cells before analysis (eg. international patentapplications WO99/66331 and WO00/09648). The device of the invention canbe used to determine the relative numbers of motile and non-motile cellsin a sample by comparing results after such a separation. The device maythus comprise means to separate motile spermatozoa from non-motilespermatozoa before introduction into the receptacle. It is not alwaysnecessary to separate cells in this way, however. For example, invasectomy verification, a test can simply indicate overall levels ofspermatozoa, motile or not.

As an alternative, the analyte may be a micro-organism. Themicro-organism might be a bacterium, such as enterotoxigenic E. coli(‘ETEC’) [e.g. see Levine (1987) J. Infect. Dis 155:377–289], for whichany suitably-labelled ETEC-specific antibody can be used as the label,such as gold-conjugated anti-CFA/I monoclonals. The micro-organism mightalso be a yeast, such as Candida.

The particulate analyte typically has a mean diameter of between 0.1 μmand 100 μm. Preferred size ranges for the analyte are 0.1–10 μm and0.1–1 μm.

Where the analyte is not naturally particulate (e.g. it is soluble), ithas to be made into particulate form in order to be assayed by thedevice of the invention. This will typically be achieved by attachingthe analyte to the surface of a small particle e.g. mono-dispersedparticles such as beads, liposomes, microparticles, microspheres,aggregates, etc. Preferred small particles are polymeric beads orparticles, such as latex or polystyrene beads. The small particle willbe coated with a receptor for the analyte, such as an immobilizedantibody. The receptor-coated particle is then mixed with a sample suchthat analyte in the sample can bind to the particle via the receptors.This gives a particle-receptor-analyte complex that is a particulateanalyte suitable for assay according to the present invention.

Preferred analytes that can be made into particulate form for assay arehormones, more preferably female hormones related to fertility such asFSH, LH, hCG etc.

The Label

The label is typically an antibody which can bind to the analyte ofinterest, and which has been suitably tagged. The tag is preferablyvisible to the naked eye (e.g., a fluorescent or colored tag), and ispreferably particulate (e.g. colloidal gold, which is visible as a pinkcolor). It will be appreciated that the term ‘antibody’ may includepolyclonal and monoclonal antibodies, as well as antibody fragments (eg.F(ab)₂, Fc etc.), sFv's etc. provided that the necessary biologicalspecificity is retained. As an alternative, the label may be a stainsuch as eosin.

The label is contained within the receptacle. The label is preferably indried form, such that it is re-constituted by addition of the liquidsample. Label may be attached to the receptacle (e.g. by spotting thelabel in liquid form, followed by drying) or may be free within thereceptacle (e.g. a pellet of freeze-dried label).

The Device

The device may include a downstream internal reference line comprising areagent which can immobilize label which was not retained by thedetection material. Comparison of the amount of label bound by thedetection material with the amount bound by the reference line allowssemi-quantitative or quantitative results to be measured.

The device of the invention can be produced simply and cheaply,conveniently in the form of a test strip or dipstick. Furthermore, itcan be used very easily, for instance by the home user. The inventionthus provides an assay device which can be used at home as a basicscreen of, for instance, male fertility.

By appropriately employing particulate antibody or antigen and labelledbinding partner, it will be appreciated that the device may be adaptedto a competitive format, as is known in the art.

The invention also provides a kit comprising (a) a receptacle containinga label and (b) a porous detection material. The kit may be used toassemble a device of the invention, or may be used by adding sample tothe receptacle, thereby activating the label, and then inserting thedetection material into the sample/label mixture.

Processes

The invention provides a process for assaying a particulate analyte,comprising the steps of:

providing a receptacle that contains (i) a label for the particulateanalyte and (ii) a porous detection material that is permeable to thelabel but is impermeable to the particulate analyte, the detectionmaterial and the label not being in liquid communication;

adding a suspension of the particulate analyte to the receptacle,thereby creating a liquid communication between the detection materialand the label;

allowing liquid in the suspension to flow into the detection materialsuch that the particulate analyte is captured by the detection material;and

detecting the interaction between the label and the captured analyte.

Where the analyte is not naturally particulate, the process preferablycomprises the initial steps of mixing the non-particulate analyte with aparticle that is coated with a receptor for the analyte, in order toform a particle-receptor-analyte complex.

EXAMPLE 1

A semen sample was separated using sodium hyaluronate at 0.88 mg/ml(Anika, Woburn, Mass.) diluted in Earle's Balanced Salt solution (GibcoBRL, Life Technologies, Scotland) containing 0.45% Bovine serum albumin(Intergen, N.Y.) and 10 mM HEPES buffer (Sigma, St. Louis, Mo.).

The number of sperm in the separated sample was counted as 30 millionper ml. Aliquots were taken and serially diluted in EBSS to give fivesamples containing 30, 15, 7.5, 3.75 and 1.9 million sperm per ml.

The assaying device (1) shown in FIG. 1 comprises a plastic receptacle(2). The bottom of the receptacle (2) has a small patch of precipitatedlabel (3), comprising monoclonal anti-CD59 labelling antibody conjugatedto 40 nm gold OD 10.0 (BRIC 229 clone, IGBRL, Bristol, UK). Withinreceptacle (2) is a 1 cm strip of SRHF nitrocellulose (porous strip) (4)(Millipore Corporation, Bedford, Mass.; high flow membrane SPHF04020),to which is attached a 3 cm wick (6) of absorbent chromatography paper(Whatman, Maidstone, UK).

50 μl of each of the five samples (5) was added to receptacle (2)together with 7.5 μl running buffer (0.5% Triton X-100+5% glucosedissolved in water). The liquid in samples (5) began to migrate throughmaterial (4) by capillary action. At the same time, label (3) wasre-constituted.

Whilst the liquid in sample (5) can enter material (4) and flow throughit, the spermatozoa in the sample are too large. Rather than enter thematerial (4), their progress is retarded to form a zone (7) ofimmobilized spermatozoa. After a short period, the re-constitutedgold-labelled antibody (3) begins to migrate through material (4),passing and visibly labelling zone (7).

An easily visible signal at zone (7) was observed within 20 minutes forthe sample containing 3.75 million sperm per ml.

EXAMPLE 2

As an alternative to the device of example 1, the device illustrated inFIG. 2 was assembled. This receptacle (2) of the device has an open end(8) remote from porous strip (4) and can thus be inserted into a sample(5).

To make receptacle (2), two acetate sheets were placed next to eachother. These were placed around strip (4), but close enough to eachother to allow capillary flow to occur between them. On one of thesheets, 7.511 of gold-labelled anti-CD59 was spotted and dried at 37° C.for 1 hour to form label (3). The sides of the receptacle were sealedwith sellotape™.

This device was dipped into samples (5) as assayed in example 1.Conjugated antibody was rapidly re-hydrated from the acetate (2–3minutes) and signal was visible at the interface between the acetate andnitrocellulose membrane at 7.5 million sperm per ml.

EXAMPLE 3

The device of example 1 was adapted to include gold-labelled anti-hCG inpatch (3).

Polystyrene particles (Sigma, LB30), 3 μm diameter, were diluted from10% to 1% solids in borate buffer (pH 8.5, 10 mM). The particles werewashed by centrifugation at 3000 rpm for 5 minutes and the supernatantwas replaced by an equal volume of fresh borate buffer.

Monoclonal anti-hCG at 5 mg/ml was added to the latex particles to givea final concentration of 150 μg/ml. The suspension was mixed for 1 hourat 20° C. before the addition of BSA to a concentration of 0.02% (w/v).The suspension was mixed for a further 30 minutes at 20° C.

The suspension was centrifuged at 300 rpm for 5 minutes and thesupernatant discarded. The pellet was re-suspended in borate buffer (pH8.5, 10 mM) to give a concentration of 1% (w/v), then washed twice, andeach time re-suspended to 1% solids in borate buffer (pH 8.5, 10 mM).

A female urine sample (containing hCG) was mixed with theantibody-conjugated particles to give a sample (5) of particulate hCGanalyte. This was added to device (1) and, after re-constitution oflabel (3) and capillary migration through detection material (4), a pinkline (7) was visible.

EXAMPLE 4

The device illustrated in FIGS. 3 and 4 was assembled. In the assembleddevice (1), tubular portion (9) receives a sample containing aparticulate analyte (e.g. a sample of motile spermatozoa). Tubularportion (9) may initially be closed (e.g., it may be filled with aplunger which, when withdrawn, fills tubular portion (9) in the mannerof a syringe). Liquid flows through tube (9) into the capillary space(2) between clear plastic housings (8 a; 8 b) and passes under a pad (3)containing dehydrated gold-tagged murine anti-CD59. As liquid passes pad(3), the antibody is re-hydrated and can pass into the liquid, where itis able to bind to spermatozoa. The liquid continues to flow towards andinto nitrocellulose (porous strip) strip (4), aided by a wick (6). Thepore size of strip (4) is too small to allow the spermatozoa to enter,so they are captured at its entrance (7). Antibody can bind capturedspermatozoa at entrance (7) and form a pink line.

FURTHER EMBODIMENTS

It will be understood that the invention is described above by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

1. A device for assaying a particulate analyte, comprising: a receptaclefor receiving a suspension of the particulate analyte, said receptaclecontaining a label for binding to the particulate analyte; and a porousdetection material having a pore size permeable to the label andimpermeable to the particulate analyte so as to substantially preventthe particulate analyte from entering into and flowing through saidporous detection material, said detection material being arranged withinsaid receptacle to allow, when the suspension is introduced into saidreceptacle, liquid in the suspension to flow into and through saiddetection material so as to capture the particulate analyte and allowthe label and the particulate analyte to interact such that the label isbound to the particulate analyte.
 2. The device of claim 1, wherein saiddetection material has a nominal pore size in a range of 20 μm to 35 μm.3. The device of claim 2, wherein said label comprises an antibody. 4.The device of claim 2, wherein said label is tagged with colloidal gold.5. The device of claim 1, wherein said detection material has a nominalpore size in a range of 5 μm to 8 μm.
 6. The device of claim 5, whereinsaid detection material comprises nitrocellulose.
 7. The device of claim6, wherein said device is operable to assay a biological cell.
 8. Thedevice of claim 6, wherein said label comprises an antibody.
 9. Thedevice of claim 6, wherein said label is tagged with colloidal gold. 10.The device of claim 5, wherein said device is operable to assay abiological cell.
 11. The device of claim 10, wherein said device isoperable to assay spermatozoa.
 12. The device of claim 11, wherein saidlabel is operable to recognize CD59.
 13. The device of claim 12, whereinsaid label comprises an antibody.
 14. The device of claim 12, whereinsaid label is tagged with colloidal gold.
 15. The device of claim 11,wherein said label comprises an antibody.
 16. The device of claim 11,wherein said label is tagged with colloidal gold.
 17. The device ofclaim 10, wherein said label comprises an antibody.
 18. The device ofclaim 10, wherein said label is tagged with colloidal gold.
 19. Thedevice of claim 5, wherein said label comprises an antibody.
 20. Thedevice of claim 5, wherein said label is tagged with colloidal gold. 21.The device of claim 1, wherein said label comprises an antibody.
 22. Thedevice of claim 21, wherein said label is tagged with colloidal gold.23. The device of claim 1, wherein said label is tagged with colloidalgold.
 24. The device of claim 1, wherein said detection material has afibrous matrix at a location whereat the particulate analyte is tocontact said detection material, said fibrous matrix having a structurefor substantially preventing the particulate analyte from enteringtherein.
 25. A process of assaying a particulate analyte, comprising:providing a receptacle containing: a label for binding to theparticulate analyte; and a porous detection material having a pore sizepermeable to the label and impermeable to the particulate analyte so asto substantially prevent the particulate analyte from entering into andflowing through the porous detection material, the detection materialand the label not being in liquid communication; adding a suspension ofthe particulate analyte to the receptacle, thereby creating liquidcommunication between the detection material and the label; allowingliquid in the suspension to flow into and through the detection materialsuch that the particulate analyte is captured by the detection material,thereby allowing the label to interact with the particulate analyte bybinding to the particulate analyte; and detecting the label bound to theparticulate analyte due to the interaction between the label and thecaptured particulate analyte, thereby indicating the presence of theparticulate analyte.
 26. The process of claim 25, wherein the detectionmaterial has a fibrous matrix at a location whereat the particulateanalyte is to contact the detection material, the fibrous matrix havinga structure for substantially preventing the particulate analyte fromentering therein.
 27. The process of claim 26, wherein the particulateanalyte is mechanically captured by being collected at a surface of thefibrous matrix.